Biomedical subjects

S B Churn

Publications and source records attributed to S B Churn.

25 records · Page 2Linked to original sources

PubMed1993

Excitotoxicity affects membrane potential and calmodulin kinase II activity in cultured rat cortical neurons.

BACKGROUND AND PURPOSE: Glutamate-induced excitotoxicity has been implicated as a causative factor for selective neuronal loss in ischemia and hypoxia. Toxic exposure of neurons to glutamate results in an extended neuronal depolarization that precedes delayed neuronal death. Because both delayed neuronal death and extended neuronal depolarization are dependent on calcium, we examined the effect of glutamate exposure on extended neuronal depolarization and calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity. METHODS: Three-week-old cortical cell cultures from embryonic rats were exposed to 500 microM glutamate and 10 microM glycine or to control medium for 10 minutes. Cells were examined for neuronal toxicity, electrophysiology, and biochemical alterations. In one set of experiments, whole-cell current clamp recording was performed throughout the experiment. In a parallel experiment, cortical cultures were allowed to recover from glutamate exposure for 1 hour, at which time the cells were homogenized and CaM kinase II activity was assayed using standard techniques. RESULTS: Excitotoxic exposure to glutamate resulted in extended neuronal depolarization, which remained after removal of the glutamate. Glutamate exposure also resulted in delayed neuronal death, which was preceded by significant inhibition of CaM kinase II activity. The excitotoxic inhibition of CaM kinase II correlated with neuronal loss, was N-methyl-D-aspartate receptor-mediated, and was not due to autophosphorylation of the enzyme. CONCLUSIONS: Glutamate-induced delayed neuronal toxicity correlates with extended neuronal depolarization and inhibition of CaM kinase II activity. Because inhibition of CaM kinase II activity significantly preceded the histological loss of neurons, the data suggest that modulation of CaM kinase II activity may be involved in the cascade of events resulting in loss of calcium homeostasis and delayed neuronal death.

Animals↗

PubMed1992

Loss of type II calcium/calmodulin-dependent kinase activity correlates with stages of development of electrographic seizures in status epilepticus in rat.

Understanding the molecular basis of altered neuronal excitability in epilepsy is a major challenge in neuroscience research. The present study suggests an inverse correlation between changes in neuronal excitability in status epilepticus and the activity of type II multifunctional calcium/calmodulin-dependent kinase II (CaM kinase II), a major Ca(2+)-signal transducing system in brain. 'Continuous' hippocampal stimulation (CHS), a new model of non-convulsive limbic status epilepticus (SE), mimics the progression of electrographic changes characteristic in human SE and allows for quantitation of post-stimulus seizure severity. In the present study, hippocampus and anterior neocortex from CHS-stimulated rats and paired surgical controls were assayed for CaM kinase II activity by incorporation of radiolabeled phosphate from [gamma-32P]ATP into the 50-kDa subunit of the kinase itself (autophosphorylation). In all instances, CHS induced sustained interictal bursting and/or electrographic seizures. Decreased CaM kinase II activity was seen in all preparations from electrically stimulated hippocampus. CaM kinase II activity in CHS animals was diminished by 37% relative to controls (P less than 0.01; Student's paired t-test). The progressive intensity of the EEG discharges correlated directly with the decrement of CaM kinase II activity (P less than 0.05; Spearman's rank correlation test, n = 5). This is the first report of a dynamic modulation of a biochemical system that has been implicated in neuronal excitability in coordination with the characterized developmental stages of SE.

Animals↗

PubMed1992

Global forebrain ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II.

Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the beta- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 microM) or high (500 microM) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the beta-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.

Animals↗

PubMed1992

Global forebrain ischemia induces a posttranslational modification of multifunctional calcium- and calmodulin-dependent kinase II.

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.

Adenosine Triphosphate↗

PubMed1991

Exposure of hippocampal slices to magnesium-free medium produces epileptiform activity and simultaneously decreases calcium and calmodulin-dependent protein kinase II activity.

The effect of magnesium-free medium on electrical and CaM kinase II activity in the rat hippocampal slice was examined. Experimental slices were incubated in 2 mM Mg, then exposed to magnesium-free medium for 1 h. Control slices were concurrently run in 2 mM Mg. Slices were then frozen and CaM kinase II activity was measured in homogenates. Exposure of hippocampal slices to magnesium-free medium resulted in spontaneous epileptiform activity and a concurrent 38 +/- 5.47% decrease in CaM kinase II activity (range 38.8-75.4% of control; n = 7, P less than 0.001, paired Student's t test). The decrease in CaM kinase II activity was not reversible by treatment with protein phosphatases 1 and 2A (58.8 +/- 4.77% of control activity; range 28.6-69.7, P less than 0.01, paired Student's t-test), indicating that the decrease in CaM kinase II activity cannot be accounted for exclusively by autophosphorylation. The results demonstrate that magnesium-free medium treatment can induce spontaneous epileptiform activity and simultaneous changes in CaM kinase II activity.

Action Potentials↗

PubMed1990

Temperature modulation of ischemic neuronal death and inhibition of calcium/calmodulin-dependent protein kinase II in gerbils.

We used brief bilateral carotid artery occlusion in gerbils to examine the effects of temperature on ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity and neuronal death. In normothermic (36 degrees C) gerbils, ischemia induced a severe loss of hippocampal CA1 pyramidal neurons measured 7 days after ischemia (28.4 neurons/mm, n = 10; control density in 10 naive gerbils 262.1 neurons/mm) and a significant decrease in forebrain calcium/calmodulin-dependent protein kinase II autophosphorylation measured 2 hours after ischemia (12.9 fmol/min, n = 6; control phosphorylation in six naive gerbils 23.5 fmol/min). The effect of temperature on these indicators of ischemic damage was examined by adjusting intracerebral temperature before and during the ischemic insult. Hyperthermic (39 degrees C) gerbils showed almost complete loss of neurons in the CA1 region (3.0 neurons/mm, n = 11) and extension of neuronal death into the CA2, CA3, and CA4 regions. In addition, hyperthermia exacerbated ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity (4.2 fmol/min, n = 6). Hypothermia (32 degrees C) protected against ischemia-induced CA1 pyramidal cell damage (257.0 neurons/mm, n = 20) and inhibition of calcium/calmodulin-dependent protein kinase II activity (26.0 fmol/min, n = 6). Our results are consistent with the hypothesis that loss of calcium/calmodulin-dependent protein kinase II activity may be a critical event in the development of ischemia-induced cell death.

Animals↗

PubMed1990

Effects of ischemia on multifunctional calcium/calmodulin-dependent protein kinase type II in the gerbil.

Cerebral ischemia produces a disruption of calcium homeostasis in neurons. This may explain the extreme sensitivity of these cells to ischemic insult. Prolonged increases in calcium levels may produce irreversible damage to the cell by altering important calcium-dependent enzyme systems such as calcium/calmodulin-dependent protein kinase II. Five minutes of acute forebrain ischemia in the gerbil produced a significant decrease in calcium/calmodulin-dependent protein kinase II activity as early as 10 seconds postischemia and persisting up to 7 days after insult. Because hypothermia protects against ischemia-induced cell death in the gerbil, we examined the effect of ischemia on cell death and calcium/calmodulin-dependent protein kinase II at different intracerebral temperatures: hyperthermia (39 degrees C), normothermia (36 degrees C), and hypothermia (32 degrees C). In ischemic animals, hyperthermia produced severe loss of neurons in CA1 and moderate loss in CA3-CA4 subregions. Normothermia in ischemic animals produced severe loss of neurons in the CA1 subregion. Hypothermic ischemic animals showed no significant loss of neurons in any hippocampal region. Ischemia produced a severe decrease (17 +/- 6% of control) in calcium/calmodulin-dependent kinase II activity in hyperthermic animals, a moderate decrease (55 +/- 15% of control) in normothermic animals, and no decrease of enzyme activity in hypothermic animals. Thus, lowering and raising intracerebral temperature decreased and increased, respectively, the extent of ischemia-induced damage in the gerbil. Because ischemia-induced effects on calcium/calmodulin-dependent protein kinase II activity are rapid and long-lasting, hypothermia may protect through preservation of calcium/calmodulin-dependent protein kinase II activity.

Animals↗